Calcium transport in uterus and bone in relationship to eggshell and bone qualities in aged laying hens

Cracked eggs and brittle bones are two major issues affecting the economic benefits of laying hens at the late laying period. Eggshell and bone quality is regulated by calcium transport in the uterus and bones of the hens. There may be an interaction between bone remodeling and eggshell calcification since bone resorption is an important calcium source for eggshell calcification. This dissertation explored the alterations of uterine calcium transport and bone remodeling as well as their impact on the eggshell and bone quality of aged hens. The relationship between eggshell and bone quality, alongside the possible mechanisms contributing to the reduction of their quality, were also examined in this thesis.
The first study investigated the effects of rearing systems on eggshell quality and bone parameters as well as the expression of genes related to bone remodeling and uterine calcium transport in aged laying hens. Layers were housed in the conventional caging system (CCS) or aviary system (AVS) from 55 days of age to 95 weeks of age. The eggshell quality decreased with age. However, at 95 weeks of age, the AVS had higher eggshell mechanical properties and components compared with the CCS, which may be attributed to the upregulated uterine calcium transporter (transient receptor potential cation channel, subfamily V, member 6 (TRPV6)) in the AVS. The AVS increased the mechanical properties, total calcium per bone, and mineral measurements of humeri, which may be associated with increased expression levels of hormone receptors (vitamin D receptor (VDR), estrogen receptor alpha (ERα), and fibroblast growth factor 23 (FGF 23)). The simultaneous upregulations of bone formation (alkaline phosphatase (ALP) and osteocalcin (OCN)) and resorption (tartrate-resistant acid phosphatase (TRAP)) related genes may contribute to the unchanged femur quality of the AVS relative to the CCS. Overall, compared with the CCS, the AVS simultaneously alleviated the deterioration of eggshell and bone qualities of aged laying hens, which may be related to the changes in the expression of genes associated with bone remodeling and uterine calcium transport. Subsequent experiments were carried out within the CCS due to the more significant decline observed in eggshell and bone quality of aged hens.
The second study compared the differences in eggshell quality, bone parameters and their correlations of aged hens laid eggs with high (HBS, 44.83 ± 1.31 N) or low (LBS, 24.43 ± 0.57 N) eggshell breaking strength. Their uterine physiological characteristics and bone remodeling processes were further explored to reveal the mechanism of eggshell and bone quality reduction and their interaction. Eggshells in the LBS showed poor quality, ultrastructural deterioration, and total calcium reduction. Bone quality was negatively correlated with eggshell quality, marked with enhanced structures and increased components in the LBS. In the LBS, the mammillary knobs and effective layer grew slowly. At the initiation stage of eggshell calcification, transcriptional profiling revealed the differentially expressed genes (DEGs) were relevant to apoptosis due to the cellular calcium overload. The level or activity of apoptosis-related proteins (p62, Bax, Bcl-2, and caspase-8), TUNEL assay, and hematoxylin-eosin staining results showed increased apoptosis and tissue damage in the uterus of the LBS. Similar damages were also observed in the uterus collected at the growth stage, although few DEGs were identified at this stage. The expressions of runt-related transcription factor 2 (RUNX 2) and OCN were upregulated in the humeri of the LBS. An enlarged diameter and more structural damages of endocortical bones and decreased ash were observed in the femurs of the HBS. The lower eggshell breaking strength may be attributed to a reduced calcium transport due to uterine tissue damage, which could affect eggshell calcification and lead to a weak ultrastructure. Impaired uterine calcium transport may result in reduced femoral bone resorption and increased humeral bone formation to maintain a higher mineral and bone quality in the LBS. The improvements in calcium metabolism of aged laying hens (such as by supplementing calcitriol or quercetin) have a potential to increase eggshell and bone quality.
The third study compared the effects of dietary calcitriol or quercetin addition on eggshell and bone quality of aged laying hens. Dietary calcitriol or quercetin supplementation improved eggshell quality (breaking strength, ultrastructure, and total calcium) and femoral bone quality (stiffness, components). Both of them increased calcium retention of hens and calcium concentration in uterine fluid at the growth stage of eggshell calcification. Additionally, dietary supplementation with calcitriol or quercetin downregulated apoptosis-related gene expression (caspase 3) at the initiation stage of eggshell calcification and improved uterine morphology. They also increased the transcripts of uterine calcium transporters (initiation stage: TRPV6; growth stage: PMCA) and hormone receptors (growth stage: VDR, ERα). These changes may be beneficial for the uterine calcium transport. Moreover, dietary addition of calcitriol or quercetin declined the femoral transcript of ALP but increased that of TRAP at the growth stage of eggshell calcification, which may contribute to the skeletal calcium delivery to the uterus. However, the concurrent upregulations in bone formation (ALP and osteopontin (OPN)) and resorption (TRAP) related genes at the initiation stage promoted the recovery of femoral bone mass. By contrast, hens fed with quercetin had higher egg production and femurs with more medullary bone and lower stiffness. Overall, dietary supplementation with calcitriol or quercetin could simultaneously improve eggshell and bone quality by modulating the calcium metabolism of aged layers.
In conclusion, eggshell quality was closely associated with bone remodeling but the relationship performs differentially due to the inconsistent physiological status of laying hens under different conditions. Compared with the CCS, the AVS that provides more space for movement would simultaneously improve bone remodeling and uterine calcium transport thus alleviating the decline of bone and eggshell qualities in aged laying hens. Under the same environment, diet, and age, the lower eggshell breaking strength was related to impaired uterine calcium transport, which may result in reduced femoral bone resorption and increased humeral bone formation to maintain a higher mineral and bone quality. Dietary calcitriol or quercetin addition could simultaneously improve eggshell and bone quality by modulating calcium metabolism in aged hens. A better-quality shell was accompanied by increased uterine calcium transport and active bone resorption. The various impacts of different conditions on bone formation may be the main reason for diverse correlations between eggshell and bone quality. Additionally, in aged laying hens, the reduced eggshell quality may be related to the less bone resorption, the uterine tissue damages and the lower calcium metabolic capacity of hens, while the deteriorated bone quality may be associated with rearing systems (the opportunities of movement), the high intensity eggshell calcification and the reduced calcium metabolic capacity of hens.